Engineering mesenchymal stem cells combined with gene therapy for osteoarticular diseases. STEMGENOS € 196.214,00

none1Rheumatoid arthritis (RA) and osteoarthritis (OA) are diseases with a population prevalence of 1 and 12% respectively, characterised by cartilage degradation and synovial inflammation leading to a irreversible loss of joint function. The resulting disabilities have a major impact on health of elderly populations, requiring hospitalisation, rehabilitation, home care with important economic impact in the European Union. The novel anti-tumor necrosis factor (TNF) a therapies availabe for RA are expensive, limited to targeting the immune/inflammatory process, do not cure RA and 20% of patients are refractory to therapy. Today there is no effective platform to regenerate articular cartilage in RA or OA. Our working hypothesis is that: "Normal joint function coul be restored effectively in RA and OA by administering adult mesenchymal stem cells (MSCs) genetically engineered to: a. suppress local inflammation and b. capable of differentiate to chondrocytes and repair the damaged cartilage". In order to validate our hypothesis we will carry out the following specific aims: Specific Aim 1: Cartilage repair in OA through engineered adult MSCs Adult MSCs will be obtained and selected from human bone marrow and murine cell lines (C3H10T1/2). In order to differentiate of Sox 9 transcription factor responsible for cartilage differentiation; 2. Use of chimeric receptor to couple collagen type II recognition to fibroblast growth factor receptor (FGFR) signalling, to induce a collagen type II-dependent Sox 9 gene expression, using an extracellular domain of an antibody to collagen type II fused to the cytoplasmic domain of the FGFR. 3. Combination of a non-signalling chimeric receptor for joint targeting and Sox 9 regulated expression. 4. Use of non-signalling chimeric receptor and treatment with engineered tarns-morphogenic transcription factor Sox-9 using recombinant proteins. Engineered MSCs will be characterized in vitro for expression of chondrocyte genes and their capacity to differentiate into cartilage cells in micropellet culture and in three dimensional scaffolds composed of polymers and hyaluronic acid. Therapeutic potential of engineered adult MSCs will be evaluated in animal models of OA (STR/ort-mice) assessing the biomechanical properties of the newly formed cartilage. Specific Aim 2: Immunotherapy in murine arthritis models through in vivo gene therapy. In vivo muscle and intraarticular electroporation of plasmids encoding for dimeric TNF receptor (dTNFR) or transforming growth factor (TGF)b, alone or in combination, under the control of autoregulated tetracycline system, will be evaluated in collagen induced arthritis (CIA), SCID mice engrafted with human cartilage and synovial tissue, huTNF transgenic mice and in the STR/or mouse OA model. Specific Aim 3: Cartilage repair combining immunotherapy in arthritis. The engineered adult MSCs will be further modified to express dTNFR and or TGFb in order to combine anti-inflammatory therapy to cartilage repair in arthritis models. Inhibition of joint inflammation will he assessed by clinical score, immunohistology, cytokine profile expression and X-ray. Simultaneously, cartilage repair will be evaluated in this model by histology, MRI and biomechanical compliance testing.noneFacchini A.Facchini A